In a medical field, a bioprocess field, and a regenerative medical field, which use cells, it is important to develop a technique of effectively and efficiently separating such cells. In addition to the centrifugation technique that has conventionally constituted mainstream, the development of a membrane material used for separation directed towards the efficiency of cell separation (reduction in the time required for filtration operations and cost reduction), and the development of a precise cell separation technique using-magnetic beads modified with antibodies, have actively been progressing in recent years. Among others, cell separation using a membrane material is considered to be an efficient cell separation technique, and thus such cell separation technique intends to be widely applied in a medical field and a bioprocess field.
For example, in a pharmaceutical manufacturing field, the development of a bioprocess of producing value-added pharmaceutical products such as erythropoietin, human growth hormone, human insulin or interferon has vigorously been progressing, using animal cells. In addition, in a bioprocess involving a suspended cell culture method for example, various membrane materials are used in a “cell separation process” of separating cells cultured at a large scale in a fermenter from a medium containing useful components, and also in a “purification process” of isolating such useful components from the separated medium. The cost required for such a cell separation process and a purification process in the aforementioned bioprocess makes up a major part in the total pharmaceutical manufacturing cost. Thus, the development of efficient cell separation and purification processes is important. Accordingly, it is strongly desired that a filter structure capable of efficiently avoiding clogging caused by cells, cell-derived components, culture solution-derived components, etc. over the whole process, and a filtration system, be designed.
In the aforementioned process using a filter, namely, in a process ranging from a cell separation process to a process of purifying useful components, in order to suppress clogging as much as possible, so as to improve cost performance regarding pharmaceutical manufacturing, first of all, it is important that cells are separated from a medium effectively (the improvement of a cell concentration rate) and efficiently (reduction in filtration time) at the initial stage. For such purpose, the effectiveness of the use of a suitable pre-filter has been known. Thus, meshes having a pore size suitable for cell separation, high pore size uniformity (a membrane material with high pore size uniformity does not have a small pore size portion that is likely to cause clogging), and high opening ratio, are often used as such pre-filters. Accordingly, it can be said that a filter having a pore size and pore size uniformity necessary for efficiently capturing and separating cells, and high opening ratio that enables rapid filtration, is preferable also in cell separation.
When a filter for separating or removing cells (generally having a size ranging from several microns to several tens of microns) is conceived, if taking into consideration deformation of cells occurring during filtration, a pre-filter having a pore size of several microns, pore size uniformity, and high opening ratio, is mainly preferably used. However, in the case of a polymeric fiber mesh filter that is generally often used as a pre-filter for example, the smallest pore size of such a filter is a square pore with a side of 20 μm or less. Thus, when such a filter is used as a filter for cell separation, cells are dropped out of such pores, and thus it does not function as an effective filter (or pre-filter) in many cases.
On the other hand, some metal mesh filters or polymeric fiber mesh filters produced by special production methods have a pore size of several μm. In such a case, the diameter of such a metal fiber or polymeric filter cannot be extremely decreased, and as a result, the opening ratio thereof is generally significantly reduced. Moreover, since water permeability is low and the clogging of the filter easily takes place, such a metal mesh filter or polymeric fiber mesh filter cannot be an effective filter used for cell separation or elimination.
As a product having a shape that is not that of a mesh filter, an “etched membrane,” which is produced by applying electron beam or ion beam to a thin membrane such as polycarbonate and then subjecting to an etching process, has been broadly known. Such an etched membrane has cylindrical pores with a uniform pore size, and the uniformity of such a pore size is extremely high. However, only several % of opening ratio can be obtained due to the production process thereof. (If opening ratio intended to increase, pore size uniformity would be lost.) When such an etched membrane is used as a separation membrane, the membrane thickness thereof must be at the minimum 10 μm for strength retention (in general, a thickness between 15 and 20 μm). However, since the pore length becomes greater than the pore size, filtration resistance increases, and thus it cannot be said that filtration efficiency is sufficient. Moreover, since cylindrical pores produced by such a production method are of straight pore-type, and many of them independently exist, it is extremely rare that pores are connected with one another in a membrane (pores communicate with one another). This is also a factor for low filtration efficiency.
Patent Document 1 discloses a porous polymer membrane wherein micro-porous layer (A) and porous layer (B) having straight pore-type pores exist as a laminated structure. This publication describes that porous layer (A) has mechanical strength and that as a result, the thickness of an etched membrane as porous layer (B) can be reduced, such as one with a thickness of 10 μm or less. However, since the opening ratio, porous structure, and internal membrane structure of porous layer (B) are the same as those of the conventional etched membrane, filtration efficiency is not sufficient. Thus, this porous polymer membrane cannot be an efficient separation filter for cells or the like.
In addition, Patent Document 2 discloses a method for producing a porous membrane with controlled pore size, which comprises applying visible ray or far ultraviolet ray to a polymer membrane via a mask and then eliminating the light irradiation region from the polymer membrane. This publication also discloses a method for forming (integrating) such a porous membrane on (with) a substrate (supporting medium) such as a non-woven fabric or synthetic paper. A porous membrane integrated with a substrate that is obtained by this method has a structure similar to that of the porous polymer membrane disclosed in Patent Document 1. However, since the region in which pores are formed can be controlled with a mask, it becomes possible to set opening ratio at relatively high. Thus, it is anticipated that filtration efficiency is also improved to a certain extent. However, since pores are formed by light irradiation, the pore structure and internal membrane structure thereof are the same as those of an etched membrane. Furthermore, this integration method involves spin-coating a polymer solution on a substrate with a rubber roller, and then drying it, so as to form a porous membrane. Thus, reduction in membrane thickness is difficult, and the polymer solution is likely to penetrate into the supporting medium substrate. As a result, reduction in the thickness of a porous membrane and the uniformity of membrane thickness become difficult, and the structure of a composite membrane is likely to become non-uniform. In particular, this phenomenon significantly takes place when a membrane is formed on a supporting porous substrate having a large average pore size, which has low filtration resistance or on which cells can easily move. Accordingly, a membrane material obtained by this technique cannot either be an efficient separation filter for cells or the like.
That is to say, a filter material having pores with a pore size of several μm (for example, approximately 1 to 5 μm), which has high pore size uniformity and high opening ratio, wherein the pores are short in the direction of a membrane thickness (that is, the membrane thickness is thin), and they are connected with one another in the membrane, and which also has excellent mechanical strength, is useful as an effective and efficient separation filter used for cells or the like (or a pre-filter).
Recently, Non-Patent Documents 1 and 2 have described that a micro droplet of water condensed and generated on a polymer solution due to the loss of latent heat during a solvent volatilization process from the polymer solution acts as a template, and that a honeycomb-structured thin porous membrane having through-pores with a pore diameter of several μm order, which has high pore size uniformity and high opening ratio, can be finally produced using various materials. This thin membrane has almost the same thickness as the diameter of a through-pore, and pores adjacent to each other are connected with each other in the membrane. Thus, this membrane adopts a structure wherein pores communicate with one another in the direction of a membrane flat surface. It is anticipated that a honeycomb-structured thin porous membrane having such a structure be used as an effective and efficient cell separation filter (or pre-filter).
Such a honeycomb-structured thin porous membrane is produced by casting a solution of an organic polymer in a hydrophobic organic solvent on a smooth solid substrate (for example, a glass, silicon wafer, metal plate, polymer solid gel, etc.), blowing air with high humidity of 40% to 95% thereon, so as to form a honeycomb structure on the substrate, and then peeling it off. However, since the strength of the obtained honeycomb-structured thin porous membrane is generally extremely low, the membrane should be peeled from the solid substrate slowly and carefully. Otherwise, the membrane is broken. Accordingly, in many cases, it is necessary that a thin membrane have previously been got wet with ethanol or the like, so as to improve peeling properties. That is to say, since the aforementioned production process using a smooth solid substrate is complicated and has poor production stability, it is naturally predicted that it becomes extremely difficult to achieve a continuous membrane formation process or a membrane formation process for realizing high productivity. In addition, in the case of using a smooth solid substrate, if the affinity of a water droplet acting as a template for the solid substrate is insufficient, a honeycomb-structured thin porous membrane has insufficient formation of through-pore in many cases. If the formation of through-pores is insufficient, it cannot exhibit functions as a filter material.
Patent Document 3 discloses a method for obtaining a honeycomb-structured thin porous membrane, which comprises casting a hydrophobic organic solvent solution on the water surface to form a honeycomb structure, and then skimming this structure with a frame of 5 mmφ. With regard to such membrane formation using a water substrate, the formation of through-pores tend to be easy. However, since it is difficult to uniformly cast a solution on the water surface, it is difficult to form a membrane with a large area. Moreover, depending on materials, wrinkles are generated due to the contraction of a membrane during a process of removing a solvent. Accordingly, it is predicted that it is extremely difficult also for this method to achieve a process for realizing high productivity.
Furthermore, a honeycomb-structured thin porous membrane material obtained by the aforementioned method has extremely low membrane strength. Thus, when such a membrane material is singly used as a cell separation filter in a bioprocess field or medical field, it is predicted that membrane break takes place at a high frequency. Further, it is also difficult to process such a membrane into a form other than a flat membrane, such as a roll-, pleated-, cylindrical, or bag-form, and to use it. That is to say, such a thin membrane material cannot directly constitute a practical filter material, and thus, it is essential to impart practical mechanical strength to such a membrane material.
A membrane material having pores with a pore diameter of several μm, which has high pore size uniformity, high opening ratio, and a membrane structure extremely excellent in terms of filtration efficiency, and which also has practical mechanical strength, is useful as an effective and efficient separation filter (or pre-filter) used for cells or the like. Such a membrane material is particularly useful for separation of blood cells in a blood filtration field, and more specifically for separation of blood plasma from whole blood or the removal of leukocytes from various blood products.
In recent years, in order to reduce the physical burden of a patient to which transfusion therapy is applied, the importance of a technique of highly removing leukocytes from a hemocyte suspension including, as typical examples, whole blood and a blood product used for transfusion (an erythrocyte product, a thrombocyte product, a blood plasma product, etc.) has increased in a medical field.
An example of a method for removing leukocytes is a filter method, which comprises filtration of a hemocyte suspension, using, as a filter element, a fibrous filter element such as a non-woven fabric, or a porous body having continuous pores in a three-dimensional network state. This filter method is advantageous in that it has high capability of removing leukocytes, in that the operations are simple and easy, and in that it is excellent in terms of cost performance. Thus, at present, the filter method is widely applied in medical sites. A filter used in this method has been known as a “leukocyte removal filter.”
Representative examples of such a leukocyte removal filter may include: filters comprising, as a filter element, a non-woven fabric consisting of ultrafine fibers such as polyester, as disclosed in Patent Documents 4 and 5; and filters comprising, as a filter element, a porous body having continuous pores in a three-dimensional network state consisting of polyurethane or the like, as disclosed in Patent Document 6. These publications disclose that the use of such filters achieves 99.99% or more of capability of removing leukocytes.
When a hemocyte suspension is filtrated using a leukocyte removal filter, a portion of the hemocyte suspension remains in a filter element after completion of the filtration. This results in the loss of a precious hemocyte suspension (in particular, in the case of an expensive blood product). Accordingly, in order to improve the cost performance of users dealing with large quantities of blood products, the need for the development of a product, the volume of a filter element of which is reduced, so as to reduce the loss of a hemocyte suspension, while maintaining the ability of a leukocyte removal filter to remove leukocytes (99.99% or more), has significantly increased under present circumstances.
The aforementioned Patent Document 4 discloses a leukocyte removal filter formed by coating the surface of a non-woven fabric used as a filter element with a coating agent containing a nonionic hydrophilic group and a nitrogen-containing basic functional group (for example, a copolymer consisting of 2-hydroxyethyl methacrylate and 2-(diethylamino)ethylmethacrylate, and then laminating a plurality of the thus coated non-woven fabrics. In this case, it is considered that the removal (capturing) of leukocytes is carried out by adsorption mechanism, and that the nitrogen-containing basic functional group has the effect of selectively adsorbing leukocytes and the nonionic hydrophilic group has the effect of suppressing non-selective adsorption of various blood cell components.
In order to reduce the volume of a filter element in such a leukocyte removal filter while maintaining its capability of removing leukocytes, it is considered adequate to increase the content of nitrogen-containing basic functional groups acting as leukocyte-selective affinity functional groups for the purpose of increasing capability of removing leukocytes per unit volume of a coated non-woven fabric. However, as a matter of fact, not only the adsorption ability of leukocytes but also the adsorption ability of other blood cell components (erythrocytes or thrombocytes) is increased by the increase in the quantities of the nitrogen-containing basic functional groups (non-selective adsorption). Consequently, the ability to selectively capture leukocytes is rather decreased. In some serious cases, the clogging of the filter occurs as a result of the adsorption of large quantities of blood cell components. Thus, it cannot be said that an increase in the quantities of nitrogen-containing basic functional groups is effective.
Patent Document 7 discloses a method for removing leukocytes from blood using an etched membrane with a pore size between 3 and 10 μm. In addition, Patent Document 8 describes that the honeycomb-structured thin porous membrane described in Non-Patent Documents 1 and 2 is used as a filter element for filtration of human blood, thereby obtaining excellent ability to selectively remove leukocytes. Interestingly, these results show that using a novel thin porous membrane material having a uniform pore size of several-μm order, depending on size effect, only leukocytes can be selectively captured from among leukocytes (with a diameter of approximately 15 μm), erythrocytes (with a diameter of approximately 7 μm), and thrombocytes (approximately 3 μm) existing in human blood. Such a filter element becomes a focus of attention also as a novel blood cell separation filter material.
However, when such an etched membrane or honeycomb-structured thin porous membrane is used as a filter element for a leukocyte removal filter, such a membrane enables only superficial capturing of leukocytes on the surface of a thin porous membrane. Thus, in order to capture all leukocytes contained in 450 cm3 of human whole blood for example, without the clogging of a filter, a thin porous membrane with an extremely large area is necessary. Consequently, since the size of a filter must be significantly larger than that of the conventional filter, such a filter is problematic in that (1) workability is significantly decreased in medical sites, (2) a filter holder (or filter housing) becomes significantly large, and the production cost is also significantly increased, and (3) in the formation of a thin porous membrane with a large area, product management (mainly, the management of pinholes or pore size uniformity) is extremely difficult in terms of mechanical strength. Thus, it is difficult to say that this is a practical technique.
As stated above, in order to significantly reduce the amount of a hemocyte suspension remaining in a filter element while maintaining the ability of a leukocyte removal filter to remove leukocytes, it is radically necessary to significantly reduce the volume of the filter element. In order to realize such reduction in the volume of filter element, it is essential to develop a leukocyte removal technique of allowing a small filter element to exhibit high capability of removing leukocytes. However, as it has conventionally been studied, it has been difficult to achieve such a technique only by designing the balance of a subtle chemical interaction between each blood cell component and a filter element surface or by optimizing functional groups.
In a medical field and a bioprocess field, in order to achieve a cell culture for allowing various types of useful cells to effectively grow, various techniques regarding the search for a culture solution composition, the design of a scaffolding for effective cell growth, etc., have been developed.
In particular, in recent years, regenerative medicine, in which stem cells having latent ability to differentiate into various types of organs are treated, has become a focus of attention. Regenerative techniques of regenerating several types of organs such as blood vessel, heart muscle, or pancreas, are at a stage in a process of clinical application. For further development of such regenerative medicine, large quantities of stem cells are necessary for conducting various basal and clinical experiments. Thus, at present, in addition to the development of a technique of collecting stem cells from a stem cell source, the development of a technique of allowing the thus collected undifferentiated stem cells to effectively grow in vitro has become a focus of attention.
For example, the effectiveness of regenerative medicine involving transplantation of hematopoietic stem cells has previously been focused in the treatments of acute myelocytic leukemia or anaplastic anemia, including bone marrow transplantation as a typical example. Currently, the effectiveness of regenerative medicine has become a focus of attention also in vascularization therapy for patients with the gravest peripheral arteriosclerosis (Buerger's disease, arteriosclerosis obliterans, diabetic gangrene, etc.). Such a vascularization therapy involving transplantation of hematopoietic stem cells has increasingly recognized by the medical profession. Accordingly, in order to further develop transplantation of hematopoietic stem cells for the treatment of various diseases including the aforementioned diseases in future, it is necessary to develop a technique of ensuring sufficient quantities of hematopoietic stem cells used for studies or clinical application.
At present, representative examples of a source of hematopoietic stem cells may include bone marrow, peripheral blood, and cord blood. From the viewpoint of noninvasiveness to a donor and reduction in hours on duty during collection of hematopoietic stem cells, at current, transplantation of hematopoietic stem cells derived from cord blood has sharply increased. For example, in April, 2003, the number of transplantation of cord blood-derived hematopoietic stem cells per month (47 cases) has exceeded the number of bone marrow transplantation (46 cases) for the first time.
However, collection of hematopoietic stem cells from a cord blood source is disadvantageous in that the amount collected from a single donor is small. Thus, under the current circumstances, cord blood-derived hematopoietic stem cells are mainly transplanted into a child patient whose body is small. Accordingly, if undifferentiated hematopoietic stem cells collected from the cord blood of a single donor were allowed to grow in vitro, the cells could also be naturally transplanted into adult patients. Thus, it can be said that this becomes an extremely revolutionary technique.
That is to say, it is important for hematopoietic stem cell transplantation to collect as many hematopoietic stem cells as possible from a single donor and then transplant them. Thus, studies have been vigorously conducted directed towards the effective growth of not only cord blood-derived hematopoietic stem cells, but also peripheral blood- and bone marrow-derived hematopoietic stem cells.
Recently, it has been reported that when cord blood-derived hematopoietic stem cells are co-cultured with mouse bone marrow-derived stromal cells in the presence of a certain kind of cytokine, the growth of undifferentiated CD34 positive cells is significantly promoted (Non-Patent Document 3). In this case, the cord blood-derived hematopoietic stem cells are co-cultured with the mouse bone marrow-derived stromal cells in a state where the two types of cells are separated from each other with a polymer diaphragm material. This publication describes that the hematopoietic stem cells are allowed to come into contact with villi extended from the stromal cells via the pores of the polymer diaphragm material, so that the above hematopoietic stem cells can effectively grow while they remain undifferentiated. If such a culture technique of co-culturing hematopoietic stem cells with different cells in a state where the two types of cells are separated with a diaphragm material and allowing the hematopoietic stem cells to grow by intracellular contact via the pores of the diaphragm were developed, it would facilitate the separation and collection of the grown hematopoietic stem cells. Accordingly, there is a possibility that such co-culture would constitute an extremely practical in vitro hematopoietic stem cell growth method.
As stated above, in order to allow a certain type of useful cells to grow by co-culture with different cells and then easily and efficiently recover such useful cells after the growth, it is effective to use a diaphragm material having a large number of pores. A diaphragm material used for such purpose is required to have the following properties:                (1) the diaphragm material has as large pores as possible within a range where cells do not move through the diaphragm, so as to effectively conduct only intracellular contact;        (2) in order to conduct effective intracellular contact, the diaphragm has high opening ratio;        (3) in order to conduct effective intracellular contact, the diaphragm has a small membrane thickness;        (4) it has high membrane strength that is sufficient for facilitating an operation to recover useful cells after the growth, or the like; and        (5) it can be processed into various membrane forms suitable for effective cell culture.        
In order to satisfy condition (1) above, membrane materials with high pore size uniformity are first selected. Thereafter, from among such membrane materials with a uniform pore size, a membrane material having the largest average pore size within a range where cells do not move through a diaphragm is preferably selected and used.
As described in the section regarding a cell separation or elimination filter, examples of such a membrane material with high pore size uniformity may include a polymeric fiber mesh, a metal mesh, an etched membrane, and a special thin porous membrane formed using a micro water droplet as a template.
A common polymeric fiber mesh has a large pore size. Thus, when such a polymeric fiber mesh is used as a cell culture diaphragm, general cells move through pores. In particular, such a polymeric fiber mesh cannot be used as a diaphragm used for the culture of hematopoietic stem cells having a diameter of approximately 7 μm.
Several metal meshes, or polymeric fiber meshes formed by special production methods, have a pore size of less than 10 μm. However, in general, since such meshes have significantly reduced opening ratio, they do not satisfy condition (2) above. Thus, although such meshes can be used as diaphragms, they do not enable effective intracellular contact. Accordingly, it cannot be said that such meshes are practical as diaphragm used for the co-culture of cells.
An etched membrane is used as a diaphragm in Non-Patent Document 3. However, the opening ratio of such an etched membrane is low, and thus this membrane cannot satisfy the aforementioned condition (2), as in the case of meshes. Thus, this etched membrane cannot be a practical diaphragm for the co-culture of cells. Although the membrane integrated with the supporting medium of Patent Document 2 satisfies condition (2) to a certain extent, but it is still insufficient. Moreover, since a polymer solution is applied on the supporting medium by spin-coating with a rubber roller, it is difficult to form a thin membrane. Further, since the polymer solution is likely to penetrate into the supporting medium substrate, it is also difficult to form a thin porous membrane and uniformize the membrane thickness. Thus, the structure of a composite membrane is likely to become non-uniform. Hence, since it is difficult to obtain a porous membrane as a uniformly thin membrane, such a porous membrane cannot satisfy the aforementioned condition (3). A membrane material produced by this technique cannot either be a diaphragm for efficient cell culture.
A special thin porous membrane produced using a micro water droplet as a template satisfies the aforementioned conditions (1) to (3). Thus, there is a possibility that this membrane can be used as a diaphragm material for efficient cell culture. However, since such a membrane has a membrane thickness of several microns, its strength is extremely low, and thus the membrane is easily broken. Accordingly, it is difficult to use a membrane with a large area for the purpose of culturing large quantities of cells or to process such a membrane into various forms (for example, in the form of a bag, roll, or the like) that are suitable for the growth of large quantities of cells or separation and collection of cells of interest. Therefore, such a membrane cannot satisfy the aforementioned conditions (4) and (5), and thus it cannot directly be a practical cell culture diaphragm.    [Non-Patent Document 1] Polymer Preprints, Japan, Vol. 50, No. 12 (2001), p. 2804    [Non-Patent Document 2] Polymer Preprints, Japan, Vol. 51, No. 5 (2002), p. 961    [Non-Patent Document 3] Saishin Igaku (Latest Medicine), Vol. 58, No. 1 (2003), p. 63    [Patent Document 1] JP-A-2-180625    [Patent Document 2] JP-A-56-135525    [Patent Document 3] JP-A-2001-157574    [Patent Document 4] International Publication WO87/05812    [Patent Document 5] U.S. Pat. No. 5,298,165    [Patent Document 6] JP-A-5-34337    [Patent Document 7] JP-A-54-46811    [Patent Document 8] JP-A-2003-149096